Hadziselimovic et al. Basic and Clinical Andrology (2021) 31:6 https://doi.org/10.1186/s12610-021-00124-w REVIEW ARTICLE Open Access Molecular clues in the regulation of mini‐ puberty involve neuronal DNA binding transcription factor NHLH2 Faruk Hadziselimovic1*, Gilvydas Verkauskas2 and Michael B. Stadler3,4 Abstract Gonadotropin releasing hormone agonist (GnRHa) treatment following surgery to correct cryptorchidism restores mini-puberty via endocrinological and transcriptional effects and prevents adult infertility in most cases. Several genes are important for central hypogonadotropic hypogonadism in mammals, including many that are transcribed in both the brain and testis. However, the expression of these genes in prepubertal gonads has not been studied systematically, and little is known about the effect of hormone therapy on their testicular and neuronal expression levels. In this review, we interpret histological sections, data on hormone levels, and RNA profiling data from adult normal testes compared to pre-pubertal low infertility risk (LIR) and high infertility risk (HIR) patients randomly treated with surgery in combination with GnRHa or only surgery. We organize 31 target genes relevant for idiopathic hypogonadotropic hypogonadism and cryptorchidism into five classes depending on their expression levels in HIR versus LIR samples and their response to GnRHa treatment. Nescient-helix-loop-helix 2 (NHLH2) was the only gene showing a decreased mRNA level in HIR patients and an increase after GnRHa treatment. This phenomenon may reflect a broader effect of hormone treatment on gene expression in both testicular and central nervous system tissues, which could explain why the hypothalamus-pituitary-testicular axis is permanently restored by the administration of GnRHa. Keywords: NHLH2, Cryptorchidism, Infertility, Mini‐puberty, Hypothalamus‐pituitary‐testicular axis, RNA-sequencing, Single‐cell RNA-sequencing Résumé Le traitement par l’agoniste de l’hormone de libération des gonadotrophines (GnRHa) suite à une intervention chirurgicale pour cryptorchidie rétablit la mini-puberté par des effets endocrinologiques et transcriptionnels et prévient l’infertilité adulte dans la plupart des cas. Plusieurs gènes jouent un rôle important dans l’hypogonadisme hypogonadotrope central chez les mammifères, dont certains sont transcrits à la fois dans le cerveau et les testicules. Cependant, l’expression de ces gènes dans les gonades prépubères n’a pas été étudiée systématiquement et l’effet de l’hormonothérapie sur leurs niveaux d’expression testiculaire et neuronale n’est pas (Continued on next page) * Correspondence: [email protected] 1Cryptorchidism Research Institute, Children’s Day Care Center Liestal, Liestal, Switzerland Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Hadziselimovic et al. Basic and Clinical Andrology (2021) 31:6 Page 2 of 13 (Continued from previous page) connu. Dans cette revue, nous interprétons des coupes histologiques, des données sur les taux d’hormones et des données de profilage d’ARN provenant de testicules normaux adultes et des patients prépubères à faible risque d’infertilité (LIR) et à haut risque d’infertilité (HIR) traités par chirurgie en association avec la GnRHa ou seulement la chirurgie dans le cadre d’une étude randomisée. Nous organisons 31 gènes cibles pertinents pour l’hypogonadisme hypogonadotrope idiopathique et la cryptorchidie en cinq classes en fonction de leurs niveaux d’expression dans les échantillons HIR et LIR et de leur réponse au traitement par GnRHa. Nescient-helix-loop-helix 2 (NHLH2) était l’unique gène dont le niveau d’ARNm diminue chez les patients HIR par rapport aux LIR et augmente suite au traitement par GnRHa. Ce phénomène pourrait être révélateur d’un effet généralisé du traitement hormonal sur l’expression des gènes dans les tissus testiculaires et du système nerveux central. Cela pourrait expliquer pourquoi l’axe hypothalamo-hypophyso-gonadique est définitivement rétablie par l’administration de la GnRHa. Mots-clés:: NHLH2, cryptorchidie, séquençage d’ARN, GnRHa, mini-puberté, infertilité, l'axe hypothalamo- hypophyso-gonadique Introduction role for the brain NHLH2 transcription factor in testicu- Re-activation of the hypothalamus-pituitary-gonadal axis lar cells and raises the interesting possibility that cura- occurs during the first weeks after birth and lasts approxi- tive hormone therapy not only influences gene mately 6 months. During this period, serum gonadotropin expression in the testis, but in the central nervous sys- and testicular testosterone, Insulin-like 3 Protein (INSL3) tem (CNS) as well, as NHLH2’s role in the brain is inhibin B, and Anti-Müllerian Hormone (AMH) levels in- known to be critical for hypogonadotropic hypogonad- crease [1, 2]. The differentiation of gonocytes into Ad (A ism. We also propose possible regulatory mechanisms dark) spermatogonia during mini-puberty is a highly critical for multi-tissue responses to hormone treatment. step during germ cell development [3, 4]. This process rep- resents the switch from a fetal pool of stem cells (gono- cytes) to an adult pool of stem cells (Ad spermatogonia) Genes involved in central hypogonadotropic that generates germ cells during a man’s entire adult life. hypogonadism fall into five distinct GnRHa response Normal development of Ad spermatogonia depends on classes luteinizing hormone (LH) and testosterone [5]. However, We interpreted our expression data from LIR/HIR and cryptorchid infants present with different degrees of impair- untreated/treated HIR samples for 31 genes and orga- ment of the testosterone increase during mini-puberty [6]. nized them into five classes according to their expression Patients whose mini-puberty and gonocyte differentiation patterns (Table 1). Class 1 (significantly lower in HIR are strongly impaired experience the most severe forms of versus LIR and significantly lower after HIR treatment) (FGFR2 infertility as adults [7]. Importantly, gonadotropin releasing consisted of fibroblast growth factor receptor 2 ). hormone (GnRH) treatment permanently induces the This gene is one of four FGFRs, and the protein kinase it transformation of gonocytes and undifferentiated spermato- regulates cell division, differentiation, migration, pro- gonia into Ad spermatogonia, which rescues adult fertility grammed cell death, and embryogenesis [15]. Single-cell in 86 % of high infertility risk (HIR) patients [8]. RNA sequencing (scRNA-Seq) data obtained with adult ’ In this review, we interpret previously published RNA testis samples revealed that the gene s expression peaks profile data obtained from testicular biopsies with a in a population of differentiating spermatogonia, which focus on genes that are implicated in idiopathic gonado- is consistent with its detection in biopsies from pre- tropin deficiency via genetic data [9] and additional can- pubertal testes that contain Ad spermatogonia (i.e., LIR; didate genes for isolated gonadotropin deficiency that Table 1; Fig. 1A,B) [16]. The expression pattern argues are not yet corroborated by genetic analyses [10, 11]. against a role for this gene in idiopathic hypogonadotro- Relevant samples from high and low infertility risk pic hypogonadism (IHH), which is in line with the fact (HIR/LIR) patients have been described elsewhere [12– that no currently known mutations associate this gene 14]. RNA isolation, purification, library preparation, se- with perturbed sexual development in males [11]. quencing, data analysis, and expression analysis were re- ported in a previous publication [12]. Among 31 genes, only NHLH2 shows decreased mes- Seven genes fall into Class 2 (significantly lower in senger ribonucleic acid (mRNA) levels in HIR versus HIR versus LIR but no significant change after HIR LIR patients, whereas GnRHa treatment increases treatment). These seven genes could be epigenetically si- NHLH2 mRNA. This intriguing result points to a novel lenced and may therefore be unable to respond to GnRHa treatment. All of them, except prokineticin receptor 1 Hadziselimovic et al. Basic and Clinical Andrology (2021) 31:6 Page 3 of 13 Table 1 scRNA-Seq profile of 31 genes known to be involved in idiopathic hypogonadotropic hypogonadism (IHH) as well as genes localized downstream of Nescient-helix-loop-helix 2 (NHL]H2). Expression values correspond to RPKM (Reads Per Kilobase of transcript per Million mapped reads), calculated